Stem cell therapy has the potential to dramatically change the treatment of human disease. The Yamaguchi laboratory is studying how the Wnt family of signaling molecules regulates the growth and development of stem cells during early embryogenesis and tumorigenesis. The laboratory is particularly interested in stem cell populations that form the neural and musculoskeletal cells of the mammalian trunk. Wnt signaling has profound effects on the development of these populations. In the absence of a Wnt signal, the muscles, cartilage, and bone of the trunk fail to form. Aberrant activation of Wnt signaling causes major abnormalities in musculoskeletal development and can promote colon cancer. Understanding how Wnt signaling regulates stem cell pathways may reveal new mechanisms to program stem cells to form neural and musculoskeletal cells valuable in cell based therapies or to target effective treatments for colon cancer. We are studying a unique progenitor known as the neuromesodermal progenitor (NMP) that resides in the primitive streak (PS) of the gastrulating embryo and gives rise to the spinal cord and musculoskeletal progenitors of the trunk and tail. Wnt3a is expressed in the PS where it regulates the self-renewal and differentiation of NMPs however the underlying mechanisms remain poorly understood. Wnt3a regulates cellular behavior by stabilizing beta(b)-catenin, which interacts with members of the Lef/Tcf family of DNA-binding factors to transcriptionally activate target genes. The Specific Aims of the laboratory are: 1) to understand how the fate of PS cells, and specifically NMPs, are regulated by Wnt3a, 2) to define the gene regulatory networks (GRN) that are activated by Wnt3a to control cell fate in the PS, 3) to define the molecular mechanisms of Wnt target gene transcription. We have made significant progress in achieving our goals: 1) We utilized cell specific markers and Tamoxifen-induced Cre-based lineage tracings to locate putative NMPs in specific germ layers of wildtype embryos. We provide functional evidence for NMP location primarily in the epithelial PS, and to a lesser degree in the ingressed PS. Lineage-tracing studies in Wnt3a/b-catenin signaling pathway mutants provide genetic evidence that trunk progenitors normally fated to enter the mesodermal germ layer can be redirected towards the neural lineage. These data, combined with previous PS lineage-tracing studies, support a model that epithelial anterior PS cells are Sox2+T+ multipotent NMPs and form the bulk of neural progenitors and paraxial mesoderm progenitors (PMPs) of the posterior trunk region. Finally, we find that Wnt3a/b-catenin signaling directs trunk progenitors towards PMP fates; however, our data also suggest that Wnt3a positively supports a progenitor state for both mesodermal and neural progenitors (Garriock et al., 2015). 2) We have generated genome-wide transcriptional profiles of wildtype (wt) and Wnt3a-/- PS and identified 729 differentially expressed genes (Dunty et al., 2014). Although this approach has successfully identified many new Wnt3a targets, it remains a significant challenge to identify the genes that are potential effectors of Wnt3a. We have therefore generated a series of ESCs carrying Doxycycline (Dox)-inducible epitope-tagged transgenes, to screen for Wnt target genes that regulate NMP development. Since stimulation of ES cells with Wnt3a rapidly induces PSM and NMPs, we reasoned that the overexpression of transcriptional effectors of Wnt3a, in the absence of exogenous Wnt3a, should induce these cell types. These studies have led us to focus on several interesting downstream transcription factors, including Mesogenin (Msgn1) and Sp5/Sp8. We are studying Msgn1, a bHLH transcription factor, since Msgn1-/- mutants indicate that it is required for PSM differentiation, a process known to be controlled by Wnt3a. Taking genetic and genomic approaches in mice and embryonic stem cells (ESCs), we have shown that Msgn1 alone controls PSM differentiation by directly activating the transcriptional programs that define PSM identity, epithelial- mesenchymal transition, motility and segmentation. Forced expression of Msgn1 in NMPs in vivo reduced NMP contribution to the neural tube, and dramatically expanded the unsegmented mesenchymal PSM, while also blocking somitogenesis and notochord differentiation. Expression of Msgn1 was sufficient to partially rescue PSM differentiation in Wnt3a-/- embryos, demonstrating that Msgn1 functions downstream of Wnt3a as the master regulator of PSM differentiation (Chalamalasetty et al., 2011; 2014). We are currently studying the role of Msgn1 in the suppression of neural fates, and are continuing to define the GRN that controls NMP development. Our works places the direct Wnt target gene, T/Brachyury, at the top of the network as it directly controls the expression of Msgn1 and Tbx6 while suppressing the expression of the NMP and neural determinant Sox2. We have also recently initiated a new project addressing the role of the Apela and Apelin family of poorly characterized peptide hormones in the regulation of NMP fates. We found that Apelin and the G-protein-coupled Apelin receptor are highly induced by Wnt3a during NMP formation. Preliminary experiments indicate that Apelin peptide, small molecule agonists, and genetic over expression of Apelin in ESCs promotes NMP-specific gene expression. These results suggest that Apelin potentiates Wnt signals during NMP specification however significantly more experimentation is necessary. 3) In an effort to unravel the mechanisms of Wnt target gene transcription, the laboratory has focused on the Sp1 family of Zinc-finger transcription factors. Sp5 is regulated by Wnt3a/beta-catenin signaling during gastrulation, and is expressed at multiple sites of Wnt activity, including normal and diseased embryonic and adult tissues, suggesting that Sp5 is a universal Wnt/b-catenin target gene. We have shown that Sp5 functions redundantly with the closely related family member Sp8 and that double mutants display a phenotype remarkably similar to Wnt3a mutants (Dunty et al., 2014). Indeed, our demonstration that Wnt3a, Ctnnb1 (b-catenin), Tcf1;Lef1, and Sp5/8 define a syn-phenotype group suggests that Sp5/8 are novel transducers of this stem cell signaling pathway, and that Sp5/8 function in the self-renewal and differentiation of NMPs. By combining genetic, genomic, biochemical and molecular biology approaches, we have shown that Sp5/8 are necessary to activate Wnt target genes, but depend upon b-catenin-Tcf1/Lef1 for activity. Intriguingly, Sp5/8 bind to GC boxes in Wnt target gene enhancers and bind directly to specific components of the enhanceosome to facilitate b-catenin recruitment. Given that Sp5 is itself a Wnt target gene, we propose that Sp5 functions as a signal amplifier in a feed-forward loop to robustly enhance Wnt target gene expression. The expression of Sp5 at most, if not all, sites of Wnt expression, including Wnt-regulated adult stem and cancer cells, suggests that the function of Sp5 in the enhancer may be a universal feature of Wnt-dependent gene expression in vertebrates (Kennedy et al., 2016). Recent studies demonstrate that Sp5/8 mutants display phenotypes in many other tissues including the brain, limb and Left-Right body axis that have been shown to be Wnt-dependent, consistent with a role for Sp5/8 in Wnt signaling. Finally, we have also recently demonstrated that Sp5/8 may play a role in linking the pluripotency GRN, that operates to maintain epiblast cells, to the Wnt/b-catenin signaling pathway through interactions with the pluripotency factor Pou5f1. We are currently examining the mechanisms for how Sp5 could disrupt the pluripotency GRN and co-opt Pou5f1 for activation of the NMP gene program by Wnt3a.